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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Abstract

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

Introduction

The use of EFs as a topical delivery platform has the potential to significantly reduce STIs. Currently, there are over 36 million people living with HIV, with over two million new cases of reported in 2015 alone1,2. Additionally, herpes simplex virus type 2 (HSV-2) infection affects hundreds of millions of people worldwide and has been shown to enhance the acquisition of HIV by 2 - 5 fold3. Due to this relationship between HSV-2 infection and HIV acquisition, there is significant interest in developing new active agents that provide simultaneous protection against multiple STIs. Moreover, the development of new vehicles to improve the delivery of these antiviral agents offers the potential to further enhance protective and therapeutic potency. Toward this goal, EFs have been investigated as a new delivery platform to reduce the prevalence of HIV-1 and HSV-2 infections.

During the past two decades, EFs have been extensively used in the fields of drug delivery and tissue engineering4. Often, biocompatible polymers are selected to easily translate to therapeutic applications. To fabricate polymeric EFs, the selected polymer is dissolved in an organic solvent or aqueous solution, depending on the degree of polymer hydrophobicity5. Active agents of interest are then added to the solvent or aqueous solution prior to the electrospinning process. The polymer solution is then aspirated into a syringe and slowly ejected while subject to an electrical current. This process typically results in polymer fibers with sheet or cylindrical macrostructures (Figure 1), and fiber diameters ranging from the micro- to nano-scale6. For most therapeutic applications, active agents are incorporated within the fibers during the electrospinning process and are released from the fiber via diffusion and subsequent fiber degradation. The rate of degradation or release may be altered by using different types of polymers or polymer blends to establish a desired release profile, imparting unique chemical and physical properties7, and promoting the encapsulation of virtually any compound. As such, EFs have proven beneficial to the delivery of small molecule drugs and biological agents including proteins, peptides, oligonucleotides, and growth factors6,8,9.

In the field of STI prevention, EFs have been recently used to incorporate and provide sustained- or inducible-release of antiviral agents10,11,12,13,14,15,16,17,18,19. In one of the earliest studies, pH-responsive fibers were developed to release active agents in response to environmental changes within the female reproductive tract (FRT), as an on-demand method of protection against HIV-111. Since, other studies have investigated polymer blends comprised of polyethylene oxide (PEO) and poly-L-lactic acid (PLLA), to evaluate the tunable release of antiviral and contraceptive agents for HIV-1 prevention and contraception in vitro12. Additional studies have demonstrated the feasibility of EFs to provide the following: prolonged release of small molecule antivirals14, strong and flexible mechanical properties20, 3-D delivery architectures21, inhibition of sperm penetration12, and the ability to merge with other delivery technologies13. Finally, previous work has evaluated polymeric fibers for the sustained-delivery of antiviral agents against common co-infective viruses, HSV-2 and HIV-114. In this study, polymer fibers provided complementary activity to antiviral delivery by retaining their structure for up to 1 month and providing a physical barrier to viral entry. From these results, it was observed that EFs may be used to both physically and chemically hinder virus infection.

While tunable release properties make polymeric EFs an attractive delivery platform for microbicide delivery, EFs have been developed in other applications to serve as surface-modified scaffolds7. EFs have been used to mimic the morphology of the extracellular matrix (ECM), often acting as scaffolds to improve cellular regeneration22, and enhance their utility in tissue engineering23,24. Fibers comprised of polymers such as poly-ε-caprolactone (PCL) and PLLA have been surface-modified with growth factors and proteins after electrospinning to impart ECM-like properties including increased cellular adhesion and proliferation25,26. Additionally, antimicrobial surface-modified EFs have been evaluated to prevent the growth of specific pathogenic bacteria27,28. Due to this versatility and the ability to induce biological effects, EF technology continues to expand across a variety of fields to provide multi-mechanistic functionality. Yet, despite their utility in a diversity of applications, surface-modified fibers have only recently been explored in the microbicide field29.

In parallel with the development of new delivery technologies to prevent and treat STIs, novel biological therapeutics have been developed. One of the most promising microbicide candidates is the adhesive antiviral lectin, GRFT30. Originally derived from a species of red algae, GRFT has demonstrated activity as a potent inhibitor of HIV, HSV-2, SARS, as well as Hepatitis C virus31,32,33,34,35,36. In fact, among biologically-based inhibitors, GRFT has the most potent anti-HIV activity, inactivating HIV-1 almost immediately upon contact30, while maintaining stability and activity in the presence of culture media from vaginal microbes for up to 10 days37. More recently, a 0.1% GRFT gel was shown to protect mice against intravaginal HSV-2 challenge, making it a promising candidate for the first line of protection against both HSV-2 and HIV-132,38. For HIV specifically, GRFT inhibits infection by physically binding gp120 or terminal mannose N-linked glycan residues on viral envelope surfaces to prevent entry38,39,40,41,42. This inhibition is highly potent, with IC50s approaching 3 ng/mL43. In addition to inhibiting HIV infection, studies have also shown that GRFT protects against HSV-2 infection by inhibiting the cell-to-cell spread of the virus32. In all cases, GRFT has been shown to be adhesive to viral particles, while demonstrating high resistance to denaturation. Last, GRFT has demonstrated synergistic activity with combinations of Tenofovir (TFV) and other antivirals44, making it feasible and likely beneficial to co-administer with EFs. The potent properties of GRFT make it an excellent biologically-based antiviral candidate, in which delivery may be enhanced with EF technology.

Utilizing this knowledge of the adhesive and innate antiviral properties of GRFT, a polymeric fiber scaffold was designed, that integrates these properties to provide the first layer of virus entry inhibition29. Finding inspiration in the way that cervicovaginal mucus hinders virus transport primarily through mucoadhesive mucin interactions, we hypothesized that by using EFs as a scaffold and covalently modifying the surface with GRFT, a high density of surface-conjugated GRFT would debilitate and inactivate virus at its entrypoint45,46,47. Here EFs were developed as a stationary scaffold to provide a protein-based, viral adhesive-inactivating barrier platform. We sought to combine the potent antiviral properties of GRFT with a biocompatible, modifiable, and durable polymer platform, to create a novel virus "trap."

To achieve these goals, fibers comprised of PLGA were electrospun, and EDC-NHS chemistry was used to subsequently modify the EF surface with GRFT. PLGA served as a model polymer due to its extensive use in electrospinning48, combined with its biocompatibility and cost-effectiveness. Additionally, surface modification exploits the large surface area of EFs, and provides a useful alternative that can be combined with encapsulation to maximize fiber utility49. Unlike traditional encapsulation methods where only a portion of GRFT is available (and only transiently present in the FRT), surface modification may enable GRFT to maintain maximum bioactivity during the entire duration of treatment. Furthermore, the incorporation of hydrophilic compounds such as proteins, by traditional electrospinning methods, may result in lower encapsulation efficiencies and loss of protein activity50. Therefore, GRFT surface-modified fibers may offer a promising alternative delivery method that can be used alone or in combination with electrospinning to enhance protection against STI infection.

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Protocol

1. Preparation and Fabrication of the Electrospun Fiber Scaffold

CAUTION: All work with solvents or polymer solutions should be performed in a chemical fume hood. Refer to material safety datasheet of each reagent before starting the protocol.

  1. To electrospin a 3 mL 15% w/w PLGA polymer solution, weigh 720 mg of 50:50 poly(lactic-co-glycolic acid) (PLGA; 0.55 to 0.75 dL/g, 31-57 kDa) into a 10 mL scintillation vial. The volume of the solution is based on the typical batch size used in current studies.
    NOTE: The polymer mass to add to a given volume of solvent must be calculated by first determining the density of the solvent used to dissolve the polymer. The density of the solvent Hexafluoro-2-propanol (HFIP) is 1.59 g/mL. Thus, the weight of solvent, based on a volume of 3.0 mL HFIP needed, is 4.8 g (3.0 mL x 1.59 g/mL). For a 15% w/w fraction of PLGA to HFIP, 720 mg PLGA must be added to 3.0 mL HFIP (0.15 x 4,800 mg = 720 mg). The advantage of using a % w/w polymer/solution, rather than % w/v, is that this provides a defined weight of the final solution. This defined weight enables more accurate solvent replacement, in the case of solvent evaporation during step 1.3.
  2. Add 3.0 mL HFIP to the glass scintillation vial containing PLGA (from step 1.1) using a serological glass pipette. Cover the vial with plastic film, then measure and record the vial mass.
  3. Incubate the polymer suspension overnight at 37 °C to ensure complete dissolution of the polymer. If any solvent evaporates, decreasing the vial mass, add HFIP until the vial reaches its original mass in step 1.2.
  4. After incubation, prepare the electrospinning apparatus (Figure 2A). Although a mandrel of any size may be used, here a rotating 25 mm outer-diameter stainless steel mandrel was used as the collector.
    NOTE: A larger mandrel diameter will decrease the fiber thickness, given the same volume of electrospinning solution.
  5. Aspirate the polymer solution into a 3 mL syringe.
  6. Connect a blunt 18-gauge, ½ inch needle tip to the syringe and dispense the excess solution (typically 0.25 mL) to remove empty headspace in the needle tip.
  7. Place the syringe on a syringe pump and set the instrument flow rate to 2.0 mL/h.
    NOTE: This flow rate was previously optimized based on polymer viscosity for this formulation.
  8. Connect the power source to the syringe needle and electrospin the polymer solution using a voltage of +27 kV. The distance between the needle and collector should be set to approximately 25 cm (Figure 2B).
    CAUTION: The electrospinning process creates a solvent vapor. Use a fume hood or an enclosed apparatus (Figure 2) to remove the harmful vapor.
  9. Once the entire solution is electrospun, turn off the power source and allow the mandrel to spin for an additional 30 min to fully evaporate solvent.
  10. Turn off the rotating mandrel collector, and use a razor blade to cut the fiber from the mandrel. Use the blade to gently peel the fiber from the mandrel.
  11. Collect the electrospun PLGA fiber into a labeled Petri dish, and place in a desiccatorovernight to remove residual solvent.

2. Surface-modification of Fibers with GRFT

  1. Prepare solutions of phosphate-buffered saline (PBS) and 2-(N-morpholino)ethanesulfonic acid (MES buffer). Prepare PBS by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 1 L of ultrapure water. Similarly, dissolve 19.52 g MES (free acid, MW 195.2) and 29.22 g NaCl in 1 L of ultrapure water, to prepare MES buffer. Ensure the final pH of each solution is between 7.2 - 7.5 and 5.0 - 6.0, respectively, using a pH meter.
  2. Prepare individual working solutions of EDC (2 mM) and NHS (5 mM).
    1. Remove the EDC and NHS from the freezer and allow them to equilibrate to room temperature before weighing.
    2. Weigh 4 mg of EDC into a 1.5 mL microcentrifuge tube.
    3. Weigh 6 mg of NHS into another microcentrifuge tube.
    4. Add 1 mL MES buffer to each tube. Vortex both tubes vigorously to ensure the reagents are fully dissolved.
  3. Prepare a solution of hydroxylamine by weighing 70 mg into a 50 mL conical centrifuge tube.
  4. Add 20 mL PBS to the hydroxylamine and vortex to dissolve.
  5. Mass out an appropriate amount of PLGA fiber into a 15 mL conical centrifuge tube. Typically, 75 mg of fiber is used for each reaction batch.
  6. Add 8 mL of MES buffer to the 15 mL tube.
  7. Add 1 mL each of the EDC and NHS solutions prepared earlier to the tube. The final volume of the solution should be 10 mL. The final concentrations of EDC and NHS should be 0.4 mg/mL and 0.6 mg/mL, respectively.
  8. Close and seal the 15 mL tube with plastic film and place on a rotator to allow the solution to be gently inverted for 15 min at room temperature (Figure 3B). This step activates the carboxyl groups on the polymer to allow for covalent modification with the GRFT protein (Figure 3A).
  9. After activation, carefully quench the reaction by adding 14 µL of β-mercaptoethanol to the tube. Invert the tube several times to ensure complete mixing.
    CAUTION: β-mercaptoethanol is highly toxic and should only be used in a chemical fume hood.
  10. Discard the supernatant and rinse the PLGA fiber twice, with 10 mL of PBS, to remove any remaining β-mercaptoethanol.
  11. After rinsing, add an appropriate amount of GRFT stock solution to the tube. For example, a 5 nmol GRFT/mg fiber would require 6.35 µL of GRFT stock solution (from a 10 mg/mL stock) per mg of fiber. Thus a 75 mg fiber sample would require 476.25 µL of 10 mg/mL GRFT stock solution.
    NOTE: GRFT fibers with theoretical loadings of 0.05, 0.5, and 5 nmol GRFT per mg fiber were fabricated.
  12. Add enough PBS to bring the final volume to 8 mL, close and invert the tube to ensure thorough mixing.
  13. Seal the tube with plastic film and place on a rotor again, this time for 2 h.
  14. After the 2 h incubation, quench the reaction by adding 2 mL of hydroxylamine solution into the 15 mL centrifuge tube. Per manufacturer instructions, the final concentration of hydroxylamine during the quenching reaction should be 0.7 mg/mL.
  15. Mix the solution well and discard the supernatant. Rinse the surface-modified PLGA fiber twice with 10 mL ultrapure water to remove any unconjugated GRFT.
  16. Transfer the fiber to a Petri dish and place inside of a desiccator until the fiber is completely dry. Transfer the Petri dish to 4 °C for storage.

3. SEM Characterization of GRFT Surface-modified Fibers

  1. Place a strip of double-sided carbon tape on an SEM specimen mount. Label the bottom of the specimen mount with the sample identifying information using a permanent marker.
  2. Cut three samples from one surface-modified fiber and place them on separate specimen mounts. The thickness of each sample is approximately 0.5 mm.
  3. Sputter coat the samples using electron-induced particle deposition from a gold plate. Sputter coat for 90 s, at 2.4 kV.
    NOTE: The sputter coat time may vary depending on equipment parameters, including voltage and amperage.
  4. Image the samples at 8 kV with a magnification ranging from 1,000 to 5,000X.

4. Extraction of GRFT from Surface-modified Fibers

  1. Mass out 2 mg of fiber in triplicate into 1.5 mL microcentrifuge tubes.
  2. Add 1 mL dimethyl sulfoxide (DMSO) to the tube, then vortex and incubate for 1 min at room temperature to completely dissolve the fiber.
  3. After incubation, dilute a 10 µL aliquot of the DMSO-fiber solution from step 4.2, at least 100-fold in Tris-EDTA (TE) buffer (pH = 8.0).
  4. Store samples at -20 °C until loading characterization with ELISA.

5. Measuring GRFT Desorption from Fibers

  1. To assess the amount of GRFT released or desorbed from the fiber, weigh 5 - 10 mg of surface-modified fiber and place in a microcentrifuge tube. Record the fiber mass in each tube.
  2. Add 1 mL of an appropriate solution that mimics the physiological environment (e.g., PBS, TE buffer, simulated vaginal fluid (SVF), etc.) to each sample.
  3. Incubate the samples for 1 h on a rotating shaker at 200 rpm, 37 °C.
  4. After incubation, remove approximately 1 mL of TE buffer containing the desorbed GRFT from the vial, and aliquot to cluster tubes. Store at -20 °C until protein quantification.
  5. Transfer the sample to a new microcentrifuge tube, add 1 mL of fresh buffer solution to the fiber within the microcentrifuge tube, and incubate until the next time point.
  6. Typical time points used to measure release include: 1, 2, 4, 6, 8, 24, 48, 72 h, and 1 wk. Negligible desorption was observed in these studies after 4 h.

6. Quantification of GRFT Extraction and Desorption via ELISA

  1. Coat a 96-well ELISA plate with 0.1 mL of HA (10 µg/mL) per well as previously described51. Seal the plate with plastic film and incubate overnight at 4 °C (Figure 4).
  2. Remove the coating buffer, and add 0.3 mL blocking buffer (2 - 3% bovine serum albumin (BSA) in PBS with 0.05% polysorbate 20) to each well. Incubate the plate for at least 2 h at room temperature.
  3. After incubation, rinse the plate 3 times with 1x PBS with 0.1% polysorbate 20 (PBS-P). After rinsing, dispense 0.1 mL of sample, standard, or PBS as negative control into the respective wells. Incubate the plate again for 1 h at room temperature.
  4. Rinse the plate 3 times again with PBS-P. After rinsing, add 0.1 mL of primary antibody (goat anti-GRFT antiserum) into each well and incubate for at least 1 h at room temperature. Typically, the primary antibody solution is diluted by 1:10,000 in PBS.
  5. After incubating the samples with primary antibody rinse the plates again 3 times with PBS-P. Add 0.1 mL of secondary antibody (horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG) to each well and incubate for 1 h at room temperature. The secondary antibody solution is diluted by 1:10,000 in PBS.
  6. Wash the plate 3 times. Add 0.1 mL TMB 2-peroxidase substrate to each well. Monitor color development (approximately 2 min), then add 0.1 mL H2SO4 (1 N) to quench the reaction. Read plate at 450 nm on a plate reader.
  7. Average the background OD values (wells which only receive PBS), and subtract this from the experimental groups.

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Results

Fiber morphology has a significant effect on the ability of surface-modified EFs to provide protection against viruses. Although electrospinning is a convenient and straightforward procedure, non-optimized polymer formulations may result in irregular fiber morphology (Figure 5B-C). Alterations in electrospinning conditions that result in the formation of beaded or amorphous mat-like morphologies, are often caused by solvent-polymer incompatib...

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Discussion

Due to their porous structures and large surface areas, EFs have found a variety of applications in healthcare, one of which includes serving as therapeutic delivery vehicles. Drugs and other active agents can be incorporated within EFs for tunable delivery, while biologics and chemical ligands can be conjugated to the fiber surface for cell-specific targeting52 or biosensing53. Here the fabrication of GRFT surface-modified PLGA EFs, as a delivery scaffold to prevent HIV in...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

We are grateful to the Jewish Heritage Fund for Excellence for funding this research. We thank Dr. Stuart Williams II for generously providing usage of the electrospinning system. We also thank Dr. Kenneth Palmer for providing us with Griffithsin. We additionally thank Dr. Nobuyuki Matoba and his lab for training us in the GRFT ELISA work.

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Materials

NameCompanyCatalog NumberComments
Poly(Lactide-co-Glycolide) (PLGA) 50:50LactelB6013-2P
1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP)Thermo Scientific 147541000
Blunt Dispensing Needle 18g X 1/2Brico Medical SuppliesBN1815
BD 3mL Syringe Luer-lok tipVWR309657
Parafilm (plastic film)Sigma AldrichP7793
2-(N-Morpholino)ethanesulfonic acid (MES Buffer)Sigma AldrichM3671 
Sodium ChlorideSigma AldrichS7653
Potassium ChlorideSigma AldrichP9333
Sodium phosphate dibasicSigma AldrichS7907
Potassium phosphate monobasicSigma AldrichP0662
Hydroxysuccinimide (NHS)Thermo Scientific 24500
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)Thermo Scientific 22980
2-MercaptoethanolFisherBP176
Griffithsin (GRFT)Kentucky BioprocessingNAcustom made, no product number
Dimethyl SulfoxideMilipore317275
Polyethylene glycol sorbitan monolaurate (Polysorbate, Tween 20)Sigma AldrichP9416 
Tris EDTA BufferSigma Aldrich93283
Flat-Bottom Immuno Nonsterile 96-Well PlatesThermo Scientific 3355
Influenza Hemagglutinin (HA)Kentucky BioprocessingNAcustom made, no product number
Goat Anti-GRFT (Primary Antibody)Kentucky BioprocessingNAcustom made, no product number
Rabbit anti-goat IgG-HRP (Secondary Antibody)Santa Cruz2056
Sure Blue TMB Microwell Peroxidase SubstrateKPL52-00-00

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